Invasion Of Riemerella Anatipestifer To Duck Embryo Fibroblast Cells

Riemerella anatipestifer (RA) infection known as infectious serositis et al, is a common bacteriosis confronting the duck industry of the world. It has a higher incidence and mortality and mainly ocurrs on domestic ducks, ducklings,1-8weeks of age are highly susceptible, also it can infect domestic geese, turkeys, chickens, it is an intracellular bacteria.At present, study on pathogenesis of RA is still in its infancy, especially on invasion of RA to duck cells. Invasion of RA to duck cells is an important and basic data for us to better illuminate its pathogenesis. In order to understand the direct interaction between RA and DEF cells, we took primary DEF cells as cell model in vitro, and choosed serotypel of RA, which is the most common strain in our country, and then carried out a preliminary study.1The analysis of the ability of adhesion and invasion to DEF cells by RAAnalysis of adheison and invasion:RA suspensions at107cfu/ml were incubated with DEF cells to allow bacteria adhesion, then cells were lysed by addition of1%Triton X-100solution respectively at2h,5h,8h. Cells lysate were plated onto chocolate agar to count the total number of adhesion. The invasion assay was the same with adhesion assay except that100μg/ml of gentamycin (GM) was used after incubation for2h,5h,8h. The results initially demonstrated that RA was able to adhere to and enter DEF cells, and the number was time dependent.Dynamic change of intracellular RA:An invasion assay was performed as described above except that after a2h invasion period, the initial GM treatment was lengthened for3,7,10h, result showed a gradual decrease in the number of viable intracellular bacterias, which was greatly significant after7h. To analyze whether RA can leave DEF cells, we carried out extracelluar bacteria assay, results showed a certain number of viable extracellular RA could be detected, this indicated that internalized RA can leave DEF cells.Electron microscope:In order to observe the adhesion and invasion of RA to DEF cells more directly, we made samples for TEM observation after incubation of RA with DEF cells for1h,5h,10h, and the results confirmed the ability of RA to adhere to and invade DEF cells. Adhesion of RA can be found by TEM after1h of inoculation, firstly attachment of RA to DEF was found, both bacteria and cells expressed a swelling at the contiguous site, the membrane of RA highly fused with membrane of DEF cells; And the presence of RA in a membrane-bound vacuole of DEF cells was observed after5h of incubation, also some vacuoles withno bacteria were found; Both apoptosis and necrocytosis of DEF were indicated by TEM after10h of incubation, the mechanism of DEF cells’death induced by RA appear complex.2Effects of the components of both bacteria and DEF cells on adhesion and invasionEffect of cytoskeleton on invasion:To study the role of host cytoskeleton components in the invasion process, DEF monolayers were preincubated with different concentrations of cytochalasin B(Sigma) or colchicine (Sigma), respectively. The invasion was inhibited significantly, which indicated that the reorganization of microfilament and microtubule was closely required for RA’s entry into DEF cells.Effect of RA’s proteins on adhesion:To determine whether bacterial proteins play a role in the adhesion of DEF, RA were pretreated with proteinase K at different concentrations, and the adhesion ability was inhibited significantly, which demonstrated that proteins of RA were involved in its adhesion.